FY2016 has seen significant progress toward accomplishing all of the Specific Aims; some of this progress is detailed here. For Aim 1, the first project focuses on the early development of MS lesions. Previously, we studied two critical aspects of lesion development: the small veins around which white matter lesions form, and the spatiotemporal dynamics of vascular permeability as manifested in gadolinium-enhanced MRI. To understand whether the presence of a central vein may help distinguish MS lesions from their mimickers an idea that remains controversial and to which we only partially subscribe we previously developed a rapid imaging approach for clinical 3T MRI called FLAIR*. Two studies to assess the utility of FLAIR* for diagnosis and characterization of MS lesions have been published in the last year (10, 25); we found that the FLAIR* technique is able to significantly improve diagnostic confidence in a variety of settings. The technique is now in use at several centers around the world, and with the North American Imaging in MS cooperative, we are planning a multi-center clinical trial to assess whether FLAIR* allows earlier and more confident diagnosis of the disease. With respect to vascular permeability, we previously established that there are two spatiotemporal patterns in MS lesions: a centrifugal pattern, in which serum contents leak from the center of the lesion and then proceed outward, over the course of minutes to hours, to fill the entire lesion; and a centripetal pattern, in which serum contents first appear on the periphery of the lesion and then proceed inward. These findings have important implications for understanding lesion development and its association with blood-brain-barrier permeability. In further work, we described how these permeability patterns help to determine the fashion in which acute MS lesions evolve into their chronic counterparts. Specifically, we found that very early events, perhaps occurring within the first month after lesion formation, appear to determine the efficacy of tissue repair, possibly including remyelination (4). Based on this research, we have started a clinical trial to test whether corticosteroids improve lesion repair, in collaboration with our colleagues in the NINDS Neuroimmunology Clinic. Additionally under Aim 1, we have completed and published work on the evolution of inflammatory demyelinating lesions in the brains of marmoset monkeys with experimental autoimmune encephalomyelitis (EAE). We previously demonstrated that the blood-brain barrier becomes locally permeable up to four weeks prior to the onset of demyelination, and we showed that this permeability is associated with a perivascular lymphocytic and mononuclear infiltrate with parenchymal activation of microglia and astrocytes. Ongoing experiments are designed to dissect the cellular and radiological correlates of neuroprotection and lesion repair in marmoset EAE in a fashion that will have direct implications for our human studies. Preliminary results indicate that the model recapitulates MS extremely well for this purpose. Finally, we completed recruitment of asymptomatic first-degree relatives of people with MS, as well as matched healthy volunteers, as part of the first stage of the nationwide Genes and Environment in Multiple Sclerosis (GEMS) study. This is a collaboration with colleagues at the Brigham & Womens Hospital of Harvard University and the University of Pittsburgh Medical Center (NCT01353547 and NCT01617395) (29). At NIH, we characterized individuals possibly at relatively high and low risk for development of clinical MS. We have presented the results of this study at several national and international meetings, and a report has been submitted to peer review. For Aim 2, work in the past year has continued to focus on development of methodology for radiological-pathological correlation studies, particularly in the marmoset EAE model. We implemented high-resolution imaging of formalin-fixed brains using a variety of MRI approaches and developed a system to use those images to guide the histopathological analysis. This is accomplished by generating 3D-printed brain-cutting boxes that allow precise sectioning of the brain, such that small lesions observed on MRI (either in vivo or postmortem) can be localized and studied. We have demonstrated the value of this system for analyzing areas of neocortical demyelination and leptomeningeal inflammation. We have further shown its ability to analyze tiny abnormal disease foci in the marmoset model (11), which we are in the process of characterizing relative to their cellular components and for the presence or absence of heavy metals. Additional work has focused on development of a clinical trial paradigm for early-phase efficacy testing of new drugs to protect and repair brain tissue undergoing inflammatory demyelination (20). In this work, we have shown that the results of conventionally but carefully acquired MRI scans, analyzed using sophisticated statistical models, can be used to infer whether such a drug failed to achieve its desired effect. This is important because there is no current method to test such drugs in trials containing fewer than dozens or hundreds of individuals that last a year or more. Our approach holds the promise of greater efficiency and sensitivity, as it can be accomplished in 6 months or less with 20 study participants or potentially even fewer. In FY16, in collaboration with the NINDS Neuroimmunology Clinic, we continued our initial testing of one new agent, developed by our extramural collaborators. We hope to complete the initial study in FY17 and move on to efficacy testing using our new trial design. We continue to make improvements to the ways in which MS lesions are imaged (6, 9, 14, 16, 18, 21, 28), using the results of those investigations to study how lesions impact short- and long-term clinical outcomes (7, 12, 17, 22, 23, 26). We have recently reviewed our research over the past few years, in the context of related studies in MS (2, 3, 5). These contributions have also made their way into position papers of several international organizations working to harmonize the diagnosis and clinical care of people with this disease (8, 15, 27).